Immunogen comprising an HIV envelope protein, a ligand and H2 peptide

ABSTRACT

The present invention relates, in general, to an immunogen and, in particular, to an immunogen for inducing antibodies that neutralize a wide spectrum of HIV primary isolates. The invention also relates to a method of inducing anti-HIV antibodies using same.

This application claims priority from Provisional Application Nos.60/234,327, filed Sep. 22, 2000, 60/285,173, filed Apr. 23, 2001,60/323,697, filed Sep. 21, 2001 and 60/323,702, filed Sep. 21, 2001, theentire contents of which are incorporated herein by reference.

This invention was made with Government support under Grant No. A135351awarded by the National Institutes of Health. The Government has certainrights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to an immunogen and, inparticular, to an immunogen for inducing antibodies that neutralize awide spectrum of HIV primary isolates. The invention also relates to amethod of inducing anti-HIV antibodies using such an immunogen.

BACKGROUND

As the HIV epidemic continues to spread world-wide, the need for aneffective HIV vaccine remains urgent. A key obstacle to HIV vaccinedevelopment is the extraordinary variability of HIV and the rapidity andextent of HIV mutation (Wain-Hobson in The Evolutionary biology ofRetroviruses, SSB Morse Ed. Raven Press, NY, pgs 185-209 (1994)).

Myers, Korber and colleagues have analyzed HIV sequences worldwide anddivided HIV isolates into groups or clades, and provided a basis forevaluating the evolutionary relationship of individual HIV isolates toeach other (Myers et al (Eds), Human Retroviruses and AIDS (1995),Published by Theoretical Biology and Biophysics Group, T-10, Mail StopK710, Los Alamos National Laboratory, Los Alamos, N. Mex. 87545). Thedegree of variation in HIV protein regions that contain CTL and T helperepitopes has also recently been analyzed by Korber et al, and sequencevariation documented in many CTL and T helper epitopes among HIVisolates (Korber et al (Eds), HIV Molecular Immunology Database (1995),Published by Theoretical Biology and Biophysics Group, Los AlamosNational Laboratory, Los Alamos, N. Mex. 87545).

A new level of HIV variation complexity was recently reported by Hahn etal by demonstrating the frequent recombination of HIV among clades(Robinson et al, J. Mol. Evol. 40:245-259 (1995)). These authors suggestthat as many as 10% of HIV isolates are mosaics of recombination,suggesting that vaccines based on only one HIV clade will not protectimmunized subjects from mosaic HIV isolates (Robinson et al, J. Mol.Evol. 40:245-259 (1995)).

The present invention relates to an immunogen suitable for use in an HIVvaccine. The immunogen will induce broadly cross-reactive neutralizingantibodies in humans and neutralize a wide spectrum of HIV primaryisolates.

SUMMARY OF THE INVENTION

The present invention relates to an immunogen for inducing antibodiesthat neutralize a wide spectrum of HIV primary isolates. The inventionalso relates to a method of inducing anti-HIV antibodies using such animmunogen.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows CCR5 or CXCR4 co-receptor proteins (or their protein orpeptide fragments) and soluble CD4 bound to gp160.

FIG. 2 shows vesicles or liposomes containing CD4, CCR5 (or CXCR4) boundto gp160 in a vesicle, liposome or an inactivated virion.

FIG. 3 shows soluble CD4 and peptides reflective of CCR5 or CXCR4 gp120binding sites bound to gp160.

FIG. 4 shows B1Acore measurements of binding interactions betweensoluble gp120 of HIV isolate DH12 and soluble CD4.

FIG. 5 shows binding interactions between HIV 89.6, JRFL, and DH12soluble gp120 proteins and soluble CD4.

FIG. 6 shows kinetic and binding affinities of the interactions betweengp120 and CD4.

FIG. 7 shows binding of soluble CD4 to gp120-expressing membranes fromthe CHO-wt (HIV-IIIB gp160-expressing) cells.

FIG. 8 shows changes in gp160 and gp140 associated with a structure ofgp160 that corresponds with the ability to induce cell fusion.

FIG. 9 shows “freezing” of fusogenic epitopes upon addition of DP-178 orT649 peptides that are parts of the coiled coil region and that, whenadded to CD4-triggered envelope, result in prevention of fusion.

FIGS. 10A-10D show binding of CD4 and CCR5 N-terminal peptide to HIV89.6 gp140 oligomers.

FIG. 10A. Overlay of the binding curves of interactions betweenimmobilized CD4 and HIV envelope proteins (89.6 gp120 and gp140). Datashow that sCD4 bound to cleaved HIV 89.6 gp140 (c1) and to HIV 89.6gp120 proteins. The negative control (negative ctrl) was generated byinjecting 89.6 gp120 proteins over a CD8 immobilized surface. Comparedto HIV JRFL and DH12 gp120 env proteins, HIV 89.6 gp120 env bound to CD4with a relatively higher affinity (K_(d)=146 nM, 96 nM and 23 nM forJRFL, DH12 and 89.6 env respectively). These differences in K_(d) werepredominantly due to differences in the off-rate (k_(d)=8.8×10⁻⁴ s⁻¹,4.7×10⁻⁴ s⁻¹, 1.1×10⁻⁴ s⁻¹ for JRFL, DH12 and 89.6 gp120 envelopeproteins, respectively).

FIG. 10B. Binding of uncleaved CM235 gp140 (uncl) and IIIB-gp160 (uncl)oligomers to immobilized CD4. Data show that whereas uncleavedIIIB-gp160 (FIG. 10B) oligomers bound poorly to CD4, uncleaved HIV CM235gp140 (FIG. 10B) oligomers did bind sCD4. The negative control is thesame injection of IIIB-gp160 flowing over a surface immobilized withIIIB-gp160 proteins.

FIG. 10C. CD4-gp140 complexes (indicated by arrow) were first formed byinjecting soluble 89.6 gp140 oligomers over a CD4-immobilized surface.Then the CCR5 N-terminal peptide (D1) was injected to monitor itsbinding to CD4-gp140 complexes.

FIG. 10D. An overlay of the binding curves of CCR5 N-terminal peptide,D1, binding to 89.6 gp140 oligomers in the presence and absence of CD4.On a CD4-gp140 complex, the D1 peptide binds with an apparent K_(d) of280 nM (k_(a)=9×10³ M⁻¹ s⁻¹, k_(d)=2.56×10⁻³ s⁻¹). In the absence ofCD4, the D1 peptide binds constitutively to cleaved gp140 oligomers witha similar on-rate but a faster off-rate (k_(d)=1.01 s⁻¹; K_(d)=1 μM).The difference in the steady-state binding of CCR5-D1 peptide in thepresence and absence of CD4 is a quantitative effect. To ensure that allgp140 molecules were bound to CD4, cleaved gp140 proteins were capturedon a CD4 immobilized sensor surface (RU=530), and soluble 89.6 gp140proteins were directly immobilized (RU=3300) on an adjacent flow cell ofthe same sensor chip.

FIGS. 11A-11D show CD4 and CCR5 extracellular domain peptides inducebinding of the HR-2 peptides to HIV 89.6 gp140.

FIG. 11A. Binding of HR-2 peptide, DP178 following binding of 89.6 gp140oligomers to CD4 (solid line), CD4 and CCR5-D1 peptide (broken line),CCR5-D1 peptide (solid circle) and to gp140 oligomers alone (brokenlines).

FIG. 11B. Binding of the HR-2 peptide, T649QL to 89.6 gp140 oligomers(broken line), CD4-gp140 complex (open circle) and to CD4-gp140-D1complex (solid line). 89.6 gp140 proteins and CCR5-D1 peptide weresequentially injected over a sCD4 immobilized surface (see FIG. 10C).

FIG. 11C. Binding of HR-2 peptides, DP178 and a control scrambled DP178peptides to CD4-89.6 gp140 complexes.

FIG. 11D. Kinetics data for the binding of HR-2 peptides, DP178 andT649QL to 89.6 gp140 after induction with CD4 and the CCR5-D1 peptide.Rm refers to the RU bound at steady-state during the injection of thesame conc of HR-2 peptides (5 mM) over the indicated gp140 complexes.nm=rate constants could not be measured due to extremely low affinitybinding of HR-2 peptides to gp140 proteins.

FIG. 12 shows immunoprecipitation by HR-2 peptide and western blotanalysis of 89.6 gp140 envelope proteins. Biotinylated HR-2 peptide,DP178 constitutively immunoprecipitated both cleaved gp41 and uncleavedgp140 proteins. The level of immunoprecipitated gp140 and gp41 wasaugmented by sCD4. HIV 89.6 gp140 proteins were incubated with 2.5 μg ofbiotinylated DP178 in the presence (Lanes 1 and 5) or absence (Lanes 3and 7) of sCD4 (2 μg). As controls, HIV 89.6 gp140 proteins were alsoincubated with 2.5 μg of biotinylated scrambled DP178 peptide in thepresence (Lanes 2 and 6) or absence (Lanes 4 and 8) of sCD4 (2 μg). Lane9 shows gp140 and gp41 within the gp140 preparation immunoblotted withmab 7B2 (anti-gp41), while Lane 10 shows gp120 within the gp140preparation reactive with mab T8 (anti-gp120).

FIG. 13 shows the induced binding of HR-2 peptides to soluble HIV 89.6gp140 envelope.

FIGS. 14A-14F show induction of HR-2 peptide binding to 89.6 gp140 bysoluble CD4. FIG. 14A. Capture of soluble 89.6 gp140 over a CD4 (solidlines) and T8 (broken lines) immobilized surface. Roughly 3000 to 5000RU of sCD4 and T8 mab were immobilized on a CM5 sensor chip. Soluble89.6 gp140 proteins were then captured to the same level on bothsurfaces and are labeled CD4-gp140 and T8-gp140 respectively. FIGS. 14Band 14C. Binding of the 19b (FIG. 14B) and 2F5 (FIG. 14C) antibodies toCD4-gp140 (solid line) and T8-gp140 (broken line) complexes. Afterstabilization of gp140 capture, either the 19b (anti-gp120 V3) or 2F5(anti-gp41) antibodies (300 μg/ml) were injected to assess the relativeamounts of gp120 and gp41 on both surfaces. FIGS. 14D-14F. Binding ofHR-2 peptide, DP178 (FIGS. 14D), scrambled DP178 (FIG. 14E) and T649Q26L(FIG. 14E) to CD4-gp140 (solid line) or T8-gp140 (broken line) surfaces.50 μg/ml of each HR-2 peptide was injected at 30 μl/min over both CD4and T8 surfaces.

FIGS. 15A-15E show binding interactions of HR-2 peptides with CD4-gp140and CD4-gp120 complexes. FIGS. 15A and 15B. Overlay of the bindingcurves showing the interactions between HR-2 peptides, DP178 (FIG. 15A)and T649Q26L (FIG. 15B), and CD4-gp120 (broken lines) and CD4-gp140(solid lines). FIGS. 15A and 15B show the titration of the HR-2 peptideDP178 and T649Q26L between 12.5 to 100 μg/ml over CD4-gp140 (solidlines) and CD4-gp120 (broken lines) surfaces. FIG. 15C. There was nobinding of scrambled DP178 HR-2 peptide to either CD4-gp140 or T8-gp140.FIG. 15D. CD4 induced HR-2 peptide binding to gp120 envelope proteins.There was no specific binding of HR-2 peptides to the T8-gp120 complex,while the sCD4-gp120 and sCD4-gp140 surfaces did stably bind HR-2peptides. Compared to CD4-gp120, higher binding was observed withCD4-gp140. Table in FIG. 15E shows the binding rate constants anddissociation constants for the HR-2 peptides and CD4-gp120 and CD4-gp140complexes. Data are representative of 2 separate experiments. HR-2peptides were injected at 30 μl/min simultaneously over a blank, sCD4and a sCD4-gp120 or sCD4-gp140 surfaces. The curves presented showspecific binding to CD4-gp120 or CD4-gp140 and were derived aftersubtraction of binding signals from blank and sCD4 surfaces.

FIGS. 16A-16F show constitutive binding of HR-2 peptides to immobilizedgp41 and CD4 induced binding to gp140. FIGS. 16A and 16B. Binding ofDP178 (12.5 to 100 μg/ml) to immobilized ADA gp41 and CD4-gp140 (FIG.16B) surfaces. FIGS. 16C and 16D. Binding of scrambled DP178 (12.5 to100 μg/ml, FIG. 16D) to immobilized ADA gp41 and the binding of HR-2peptide, DP178 (12.5 to 50 μg/ml) to T8-gp140 surfaces. FIGS. 16E and16F. Scatchard plots of RU vs RU/C for DP178 binding to gp41 (FIG. 16E)and to CD4-gp140 (FIG. 16F). Data are representative of 2 separateexperiments.

FIGS. 17A and 17B show induction of HR-2 peptide binding to gp120-gp41complex. Binding of HR-2 peptide DP178 (50 μg/ml) to immobilized gp41,gp41-gp120 and gp41-gp120-CD4 complexes. Compared to gp41 surface,higher binding of DP178 is observed on gp41-gp120 and gp41-gp120-CD4surfaces. Soluble 89.6 gp120 (100 μg/ml) or a gp120-CD4 mixture (gp120pre-incubated with 0.3 mg/ml of CD4 for 30 min) was injected at 5 μl/minfor 10 min over a sensor surface immobilized with ADA gp41. At the endof the injections, wash buffer (PBS) was allowed to flow until thesurface was stable with no baseline drift. Roughly 300 RU of gp120 and850 RU of gp120-CD4 formed stable complexes with the immobilized gp41surface. The gp120-CD4 binding was subsequently reduced to about 325 RU(to be rougly equivalent to gp41-gp120 surface) with short injections ofa regeneration buffer (10 mM glycine-HCl, pH 2.0), followed by aninjection of gp120-CD4 mixture. Thus both surfaces were stable and hadequivalent amount of associated gp120 or gp120-CD4.

FIG. 18. Soluble CD4 binding to HIV gp120 non-covalently linked to gp41results in binding of HR-2 to gp120 and upregulation of gp41-gp120association. An immunogen is cross-linked CD4-gp120-gp41 in solubleform.

FIG. 19. A32 binding to HIV gp120 non-covalently linked to gp41 resultsin binding of HR-2 to gp120 and upregulation of gp41-gp120 association.An immunogen is cross-linked A32 (whole Ig)-gp120-gp41 in soluble formor A32 (Fab or Fab2)-gp120-gp41 in soluble form.

FIG. 20. Soluble CD4 binding to soluble gp120 results in exposure of theCCR5 binding site, and the gp41 HR-2 binding site.

FIG. 21. A32 mab binding to soluble HIV gp120 results in exposure of theCD4 binding site and the CCR5 binding site.

FIG. 22. Soluble CD4 binding to soluble uncleaved HIV gp140 upregulatesHR-2 binding to HR-1 in gp41, and exposes the CCR5 binding site. Animmunogen is crosslinked CD4-gp140 with or without DP178 or T649Q26Lbound.

FIG. 23. Mab A32 binding to soluble uncleaved HIV gp140 upregulates HR-2binding to HR-1 in gp41, and exposes the CCR5 binding site. An immunogenis crosslinked A32 or a fragment thereof—gp140 with or without DP178 orT649Q26L bound to gp41 or elsewhere in the complex.

FIG. 24. Soluble CD4 binding to HIV gp120 non-covalently linked to gp41results in binding of HR-2 to gp120 and upregulation of gp41-gp120association. An immunogen is cross-linked CD4-gp120-gp41 in solubleform, with HR-2 peptides bound to HR-1.

FIG. 25. A32 binding to HIV gp120 non-covalently linked to gp41 resultsin binding of HR-2 to gp120 and upregulation of gp41-gp120 association.An immunogen is cross-linked A32 (whole Ig)-gp120-gp41 in soluble formor A32 (Fab or Fab2)-gp120-gp41 in soluble form, with HR-2 peptide boundto HR-1 or elsewhere in the complex.

FIG. 26. Induction of HR-2 peptide binding to HIV 89.6 gp140 by solubleCD4. Soluble 89.6 gp140 proteins were captured to the same level on allthree surfaces and are labeled CD4-gp140, T8-gp140 and A32-gp140. Figureshows binding of HR-2 peptide DP178 to T8-gp140 (circle), CD4-gp140(solid line) and A32-gp140 (broken line) complexes. 50 μg/ml of HR-2peptide was injected at 30 μl/min over A32, CD4 and T8 surfaces. Onlyspecific binding, after bulk responses and non-specific binding of HR-2peptides to sCD4, T8 and A32 surfaces were subtracted, are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an immunogen that induces broadlyreactive neutralizing antibodies that are necessary for an effectiveAIDS vaccine. In one embodiment, the immunogen comprises a cleaved oruncleaved gp140 or gp160 HIV envelope protein that has been “activated”to expose intermediate conformations of conserved neutralizationepitopes that normally are only transiently or less well exposed on thesurface of the HIV virion. The immunogen is a “frozen” triggered form ofHIV envelope that makes available specific epitopes for presentation toB lymphocytes. The result of this epitope presentation is the productionof antibodies that broadly neutralize HIV.

The concept of a fusion intermediate immunogen is consistent withobservations that the gp41 HR-2 region peptide, DP178, can capture anuncoiled conformation of gp41 (Furata et al, Nature Struct. Biol. 5:276(1998)), and that formalin-fixed HIV-infected cells can generate broadlyneutralizing antibodies (LaCasse et al, Science 283:357 (1997)).Recently a monoclonal antibody against the coiled-coil region bound to aconformational determinant of gp41 in HR1 and HR2 regions of thecoiled-coil gp41 structure, but did not neutralize HIV (Jiang et al, J.Virol. 10213 (1998)). However, this latter study proved that thecoiled-coil region is available for antibody to bind if the correctantibody is generated.

Conserved neutralization sites on the HIV envelope can be on tworegions; they can be on gp41 and they can be on gp120.

The regions and conformations of gp41 that are exposed during gp140 orgp160 “triggering” (“activation”) can be expected to be conserved since:i) the amino acid sequences of the coiled-coil region are conserved andii) the function of the fusogenic envelope complex is conserved andessential for virus pathogenicity. This conservation is key to theproduction of broadly neutralizing anti-HIV antibodies.

The immunogen of one aspect of the invention comprises HIV envelopecleaved or uncleaved gp140 or gp160 either in soluble form or anchored,for example, in cell vesicles made from gp140 or gp160 expressing cells,or in liposomes containing translipid bilayer HIV gp140 or gp160envelope. Alternatively, triggered gp160 in aldrithio 1-2 inactivatedHIV-1 virions can be used as an immunogen. The gp160 can also exist as arecombinant protein either as gp160 or gp140 (gp140 is gp160 with thetransmembrane region and possibly other gp41 regions deleted). Bound togp160 or gp140 can be recombinant CCR5 or CXCR4 co-receptor proteins (ortheir extracellular domain peptide or protein fragments) or antibodiesor other ligands that bind to the CXCR4 or CCR5 binding site on gp120,and/or soluble CD4, or antibodies or other ligands that mimic thebinding actions of CD4 (FIG. 1). Alternatively, vesicles or liposomescontaining CD4, CCR5 (or CXCR4) (FIG. 2), or soluble CD4 and peptidesreflective of CCR5 or CXCR4 gp120 binding sites (FIG. 3). Alternatively,an optimal CCR5 peptide ligand can be a peptide from the N-terminus ofCCR5 wherein specific tyrosines are sulfated (Bormier et al, Proc. Natl.Acad. Sci. USA 97:5762 (2001)). The data in FIG. 13 clearly indicatethat the triggered immunogen may not need to be bound to a membrane butmay exist and be triggered in solution. Alternatively, soluble CD4(sCD4) can be replaced by an envelope (gp140 or gp160) triggered by CD4peptide mimetopes (Vitra et al, Proc. Natl. Acad. Sci. USA 96:1301(1999)). Other HIV co-receptor molecules that “trigger” the gp160 orgp140 to undergo changes associated with a structure of gp160 thatinduces cell fusion can also be used. (See also FIG. 8.) The datapresented in Example 2 demonstrate that ligation of soluble HIV gp140primary isolate HIV 89.6 envelope with soluble CD4 (sCD4) inducedconformational changes in gp41 (see FIG. 13).

In one embodiment, the invention relates to an immunogen that has thecharacteristics of a receptor (CD4)-ligated envelope with CCR5 bindingregion exposed but unlike CD4-ligated proteins that have the CD4 bindingsite blocked, this immunogen has the CD4 binding site exposed (open).Moreover, this immunogen can be devoid of host CD4, which avoids theproduction of potentially harmful anti-CD4 antibodies uponadministration to a host. (See FIGS. 18-25.)

The immunogen can comprise gp120 envelope ligated with a ligand thatbinds to a site on gp120 recognized by an A32 monoclonal antibodies(mab) (Wyatt et al, J. Virol. 69:5723 (1995), Boots et al, AIDS Res.Hum. Retro. 13:1549 (1997), Moore et al, J. Virol. 68:8350 (1994),Sullivan et al, J. Virol. 72:4694 (1998), Fouts et al, J. Virol. 71:2779(1997), Ye et al, J. Virol. 74:11955 (2000)). One A32 mab has been shownto mimic CD4 and when bound to gp120, upregulates (exposes) the CCR5binding site (Wyatt et al, J. Virol. 69:5723 (1995)). Ligation of gp120with such a ligand also upregulates the CD4 binding site and does notblock CD4 binding to gp120. Advantageously, such ligands also upregulatethe HR-2 binding site of gp41 bound to cleaved gp120, uncleaved gp140and cleaved gp41, thereby further exposing HR-2 binding sites on theseproteins—each of which are potential targets for anti-HIV neutralizingantibodies.

In a specific aspect of this embodiment, the immunogen comprises solubleHIV gp120 envelope ligated with either an intact A32 mab, a Fab2fragment of an A32 mab, or a Fab fragment of an A32 mab, with the resultthat the CD4 binding site, the CCR5 binding site and the HR-2 bindingsite on gp120 are exposed/upregulated. The immunogen can comprise gp120with an A32 mab (or fragment thereof) bound or can comprise gp120 withan A32 mab (or fragment thereof) bound and cross-linked with across-linker such as 0.3% formaldehyde or a heterobifunctionalcross-linker such as DTSSP (Pierce Chemical Company). The immunogen canalso comprise uncleaved gp140 or a mixture of uncleaved gp140, cleavedgp41 and cleaved gp120. An A32 mab (or fragment thereof) bound to gp140and/or gp120 or to gp120 non-covalently bound to gp41, results inupregulation (exposure) of HR-2 binding sites in gp41, gp120 anduncleaved gp140. Binding of an A32 mab (or fragment thereof) to gp120 orgp140 also results in upregulation of the CD4 binding site and the CCR5binding site. As with gp120 containing complexes, complexes comprisinguncleaved gp140 and an A32 mab (or fragment thereof) can be used as animmunogen uncross-linked or cross-linked with cross-linker such as 0.3%formaldehyde or DTSSP. In one embodiment, the invention relates to animmunogen comprising soluble uncleaved gp140 bound and cross linked to aFab fragment of an A32 mab, optionally bound and cross-linked to an HR-2binding protein.

The gp120 or gp140 HIV envelope protein triggered with a ligand thatbinds to the A32 mab binding site on gp120 can be administered incombination with at least a second immunogen comprising a secondenvelope, triggered by a ligand that binds to a site distinct from theA32 mab binding site, such as the CCR5 binding site recognized by mab17b. The 17b mab (Kwong et al, Nature 393:648 (1998) available from theAIDS Reference Repository, NIAID, NIH) augments sCD4 binding to gp120.This second immunogen (which can also be used alone or in combinationwith triggered immunogens other than that described above) can, forexample, comprise soluble HIV gp120 envelope ligated with either thewhole 17b mab, a Fab2 fragment of the 17b mab, or a Fab fragment of the17b mab. It will be appreciated that other CCR5 ligands, including otherantibodies (or fragments thereof), that result in the CD4 binding sitebeing exposed can be used in lieu of the 17b mab. This further immunogencan comprise gp120 with the 17b mab, or fragment thereof, (or other CCR5ligand as indicated above) bound or can comprise gp120 with the 17b mab,or fragment thereof, (or other CCR5 ligand as indicated above) bound andcross-linked with an agent such as 0.3% formaldehyde or aheterobifunctional cross-linker, such as DTSSP (Pierce chemicalCompany). Alternatively, this further immunogen can comprise uncleavedgp140 present alone or in a mixture of cleaved gp41 and cleaved gp120.Mab 17b,or fragment thereof (or other CCR5 ligand as indicated above)bound to gp140 and/or gp120 in such a mixture results in exposure of theCD4 binding region. The 17b mab, or fragment thereof, (or other CCR5ligand as indicated above)-gp140 complexes can be present uncross-linkedor cross-linked with an agent such as 0.3% formaldehyde or DTSSP.

Soluble HR-2 peptides, such as T649Q26L and DP178 (see below), can beadded to the above-described complexes to stabilize epitopes on gp120and gp41 as well as uncleaved gp140 molecules, and can be administeredeither cross-linked or uncross-linked with the complex.

A series of monoclonal antibodies (mabs) have been made that neutralizemany HIV primary isolates, including, in addition to the 17b mabdescribed above, mab IgG1b12 that binds to the CD4 binding site on gp120(Roben et al, J. Virol. 68:482 (1994), Mo et al, J. Virol. 71:6869(1997)), mab 2G12 that binds to a conformational determinant on gp120(Trkola et al, J. Virol. 70:1100 (1996)), and mab 2F5 that binds to amembrane proximal region of gp41 (Muster et al, J. Virol. 68:4031(1994)). A mixture of triggered envelope immunogens can be used tooptimize induction of antibodies that neutralize a broad spectrum of HIVprimary isolates. Such immunogens, when administered to a primate, forexample, either systemically or at a mucosal site, induce broadlyreactive neutralizing antibodies to primary HIV isolates.

As indicated above, various approaches can be used to “freeze” fusogenicepitopes in accordance with the invention. For example, “freezing” canbe effected by addition of the DP-178 or T-649Q26L peptides thatrepresent portions of the coiled coil region, and that when added toCD4-triggered envelop, result in prevention of fusion (Rimsky et al, J.Virol. 72:986-993 (1998) (see FIGS. 9 and 13). HR-2 peptide bound gp140,gp41 or gp160 can be used as an immunogen or crosslinked by a reagentsuch as DTSSP or DSP (Pierce Co.), formaldehyde or other crosslinkingagent that has a similar effect (see below).

“Freezing” can also be effected by the addition of 0.1% to 3%formaldehyde or paraformaldehyde, both protein cross-linking agents, tothe complex, to stabilize the CD4, CCR5 or CXCR4, HR-2 peptide gp160complex, or to stabilize the “triggered” gp41 molecule, or both (LaCasseet al, Science 283:357-362 (1999)).

Further, “freezing” of gp41 or gp120 fusion intermediates can beeffected by addition of heterobifunctional agents such as DSP (dithiobis[succimidylproprionate]) (Pierce Co. Rockford, Ill., No. 22585ZZ) or thewater soluble DTSSP (Pierce Co.) that use two NHS esters that arereactive with amino groups to cross link and stabilize the CD4, CCR5 orCXCR4, HR-2 peptide gp160 complex, or to stabilize the “triggered” gp41molecule, or both.

Inherent differences exist in HIV isolates among HIV clades and amongHIV isolates from patients in varying geographic locations. Triggeredcomplexes for HIV vaccine development can be made with HIV envelopesfrom a variety of HIV clades and from a variety of locations. Triggeredcomplexes comprising antibodies or fragments thereof that upregulate theCCR5 binding site, the CD4 binding site, or both, or antibodies, orfragments thereof, that are CD4 inducible can be produced byco-expressing in a dicistronic manner in a plasmid both gp120 and, forexample, the heavy and light chain of the Fab region of the antibody, inorder to produce a recombinant protein that has the properties of theabove described complexes.

The immunogen of the invention can be formulated with a pharmaceuticallyacceptable carrier and/or adjuvant (such as alum) using techniques wellknown in the art. Suitable routes of administration of the presentimmunogen include systemic (e.g. intramuscular or subcutaneous).Alternative routes can be used when an immune response is sought in amucosal immune system (e.g., intranasal).

Certain aspects of the invention are described in greater detail in thenon-limiting Example that follows.

EXAMPLE 1

The feasibility of the immunogen production approach of the presentinvention has been shown using BiaCore 3000 technology. FIG. 4 showsthat CD4, not CD8, can be bound to gp120. FIGS. 5, 6 show thedifferences in interaction of gp120 with the envelop of primary isolatesand lab-adapted HIV strains. FIG. 7 shows that vesicles from chimerichamster ovary cells transfected with HIV-IIIB gp160 and that thesevesicles, present on a BiaCore L1 Chip, show stabilized binding ofsoluble CD4 but not CD8.

EXAMPLE 2 Experimental Details Proteins

Soluble, monomeric gp120 JRFL, gp120 DH12 and sCD4 were produced byProgenics, and were provided by the Division of AIDS, NIAID, NIH.HIVIIIB gp160 was obtained from Protein Sciences. Envelope proteins fromHIV 89.6 (Clade B), and HIV CM235 (Clade E) primary isolates wereproduced by Pat Earl, NIH, using recombinant vaccinia viruses andpurified as described (Earl et al, J. Virol. 68:3015-3026 (1994), Earlet al, J. Virol. 75:645-653 (2001)).

Briefly, BS-C-1 cells in 160 cm² flasks were infected with vBD1(HIV 89.6gp140) or vBD2 (gp120) vaccinia viruses. After 2 h, the cells werewashed in PBS and placed in serum-free OPTI-MEM media (Gibco) for 24-26hr. The culture medium was then harvested by centrifugation andfiltration (0.2 μm) and then TX-100 was added to 0.5% (final conc.,v/v). For some of the experiments, the culture medium was concentrated15-30 fold and served as a source of multimeric gp140 glycoproteins (amix of cleaved and uncleaved form). Further purification of theseglycoproteins was performed using a two-step procedure. In the firststep, contaminating proteins were removed and glycoproteins from themedium were bound to a lentil lectin column and eluted with methylα-D-mannopyranoside. This preparation contained ˜50:50 cleaved anduncleaved gp140 and the per-purified culture supernatant concentrate aretermed “cleaved gp140”. Finally, oligomeric and dimeric gp140 wereseparated and purified by size exclusion chromatography. This gp140preparation is termed “uncleaved gp140”. The glycoprotein fractions werepooled and concentrated using micro-concentrators.

Monoclonal Antibodies

Human monoclonal antibody against the gp120 V3 loop (19b) the CCR5binding site (17b), and mab 7B2 against the immunodominant region ofgp41 were the gifts of James Robinson (Tulane University, New Orleans,La.). Mabs 2F5, IgG1b12 and 2G12 were obtained from the AIDS ReferencesRepository, NIAID, NIH.

CCR5 and HR-2 Peptides

Synthetic peptides were synthesized (SynPep Corporation, Dublin,Calif.), and purified by reverse phase HPLC. Peptides used in this studyhad greater than 95% purity as determined by HPLC, and confirmed to becorrect by mass spectrometry. The CCR5-D1(MDYQVSSPIYDINYYTSEPCQKINVKQIAAR) (SEQ ID NO:l), peptide was derivedfrom the N-terminus of human CCR5 (Bieniasz et al, EMBO Journal16:2599-2609 (1997)) . Gp4l peptides DP-178YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO:2) (Wild et al, Proc.Natl. Acad. Sci. USA 91:12676-12680 (1994)), T-649WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL (SEQ ID NO:3) (Rimsky et al, J.Virol. 72:986-993 (1998)), and T649-Q26L(WMEWDREINNYTSLIHSLIEESQNQLEKNEQELLEL) (SEQ ID NO:4) (Shu et al,Biochemistry 39:1634-1642 (2000)) were derived from HIV-1 envelope gp41from HIV 89.6 (Collmann et al, J. Virol. 66:7517-7521 (1992)). As acontrol for HR-2 peptide binding, a scrambled sequence DP178 peptide wasmade as well.

Surface Plasmon Resonance Biosensor Measurements

SPR biosensor measurements were determined on a BIAcore 3000 (BIAcoreInc., Uppsala, Sweden) instrument. HIV envelope proteins (gp120, gp140,gp160) and sCD4 were diluted to 100-300 mg/ml in 10 mM Na-Acetatebuffer, pH 4.5 and directly immobilized to a CM5 sensor chip usingstandard amine coupling protocol for protein immobilization (Alam et al,Nature 381:616-620 (1996)). Binding of proteins and peptides wasmonitored in real-time at 25° C. and with a continuous flow of PBS, pH7.4 at 5-20 ml/min. Analyte (proteins and peptides) were removed and thesensor surfaces were regenerated following each cycle of binding bysingle or duplicate 5-10 ml pulses of regeneration solution (10 mMglycine-HCl, pH 2.5 or 10 mM NaOH).

All analyses were performed using the non-linear fit method ofO'Shannessy et al. (O'Shannessy et al, Anal. Biochem. 205:132-136(1992)) and the BIAevaluation 3.0 software (BIAcore Inc). Rate andequilibrium constants were derived from curve fitting to the Langmuirequation for a simple bimolecular interaction (A+B=AB).

In preliminary SPR experiments, it was determined that HIV gp120envelope protein for HIV89.6 bound sCD4 most avidly with relativelylittle baseline drift (t_(1/2) of binding, 105 min.) compared to HIVgp120 DH12 (t_(1/2) of binding, 25 min) and HIV gp120 JRFL (t_(1/2) ofbinding 14 min.). Thus, HIV89.6 gp120 and gp140 were produced forsubsequent experiments.

Immunoprecipitation of HIV Envelope Proteins Followed by Western BlotAnalysis. Soluble HIV 89.6 gp140 or gp120 proteins were incubated withor without 2 μg of recombinant sCD4, and a dose range if eitherbiotinylated DP178 or biotinylated scrambled DP178 as a control in atotal volume of 50 μl PBS for 2 h followed by incubation (4 h) with 50μl suspension of streptavidin-agarose beads (Sigma Chemicals, St. Louis,Mo.). Immune complexes were washed×3 with 500 μl of PBS, resuspended inSDS-PAGE sample buffer containing 2-ME, boiled for 5 min, and loadedonto SDS-PAGE on 4-20% polyacrylamide gels. Gels were transferred toimmunoblot membranes for Western blot analysis with either mabs T8(anti-gp120 N-terminus) or 7B2 (anti-gp41 immunodominant region).

Results

Binding of sCD4 to Cleaved and Uncleaved HIV Envelope. Preliminary SPRstudies showed that, of HIV gp120 envelopes JRFL, DH12 and 89.6, the HIV89.6 gp120 demonstrated the most stable binding to sCD4 (see FIG. 10A).Thus, preparations of HIV89.6 gp140 envelope proteins were studied thatcontained either cleaved or primarily uncleaved gp140 for binding tosCD4. Non-cleaved HIV CM235 gp140 and, as well, cleaved HIV 89.6 gp140,were tested for their ability to bind sCD4 covalently anchored on thechip in the SPR biosensor assay. Cleaved 89.6 gp140 bound sCD4 well(FIG. 10A). The uncleaved baculovirus-produced HIV IIIB gp160 fromProtein Sciences did not bind sCD4 (Mascola et al, J. Infect. Dis.173:340-348 (1996)) (FIG. 10B). However, cleavage of gp140 into gp120and truncated gp41 was not an absolute requirement for binding of sCD4to envelope preparations, as the uncleaved Clade E envelope HIV CM235gp140 also bound sCD4 (FIG. 10B).

Binding of an N-terminal CCR5 Extracellular Domain Peptide to HIV 89.6gp140 Envelope. The CCR5 binding site on HIV gp120 is inducible by sCD4(Kwong et al, Nature 393:648-659 (1998), Rizzuto et al, Science280:1949-1953 (1998), Wyatt et al, Nature 393:705-710 (1998)). It wasnext determined if an N-terminal CCR5 extracellular domain syntheticpeptide could be made that bound to HIV 89.6 gp140. A 30 aa peptide wasproduced from the N-terminus of CCR5 (termed CCR5-D1), and tested forability to bind to cleaved HIV 89.6 gp140.

Low levels of constitutive binding of the CCR5-D1 peptide to cleavedgp140 were found, while sCD4 binding to cleaved gp140 envelope inducedmore stable binding of CCR5-D1 binding to gp140 (FIG. 10C and 10D). Domset al have found that the affinity of native CCR5 to sCD4 ligated gp120in the context of membrane bound envelope was 500 μM (Hoffman et al,Proc. Natl. Acad. Sci. 97:11215-11220 (2000)). The binding affinity ofthis CCR5-D1 N-terminal domain peptide to sCD4-ligated gp140 was foundto be ˜280 μM (FIG. 10D).

Effect of Soluble CD4 and the CCR5-D1 Extracellular Domain Peptide onthe Binding of HR-2 Peptides to Cleaved HIV 89.6 gp140 EnvelopeProteins. A major goal of this study was to determine iffusion-associated conformations of gp41 could be detected using SPRassays of HR-2 peptide binding. It was reasoned that if HR-2 peptidescan bind gp140, the gp41 coiled-coil structure must be uncoiled, suchthat endogenous HR-2 is not bound to HR-1. Three such HR-2 peptides wereused in this study, DP178 (Wild et al, Proc. Natl. Acad. Sci. USA91:12676-12680 (1994), Rimsky et al, J. Virol. 72:986-993 (1998)), T-649(Rimsky et al, J. Virol. 72:986-993 (1998)), and T649Q26L (Shu et al,Biochemistry 39:1634-1642 (2000)). DP178 contains C-terminal amino acidsof HR-2 (Wild et al, Proc. Natl. Acad. Sci. USA 91:12676-12680 (1994),Rimsky et al, J. Virol. 72:986-993 (1998)) while T649Q26L contains moreN-terminal amino acids of HR-2 (Rimsky et al, J. Virol. 72:986-993(1998), Shu et al, Biochemistry 39:1634-1642 (2000)). Low level bindingDP178 to cleaved HIV 89.6 gp140 was found (FIG. 11A), and DP178 controlscrambled peptide did not bind at all (FIG. 11C).

When binding of HR-2 peptides were determined on cleaved gp140 ligatedwith sCD4, DP178 HR-2 peptide binding was induced on cleaved gp140 bysCD4 (FIG. 11A). Addition of the CCR5-D1 N-terminal peptide tosCD4-gp140 complexes did not significantly change the Kd of DP178binding to gp140 (0.7 to 1.1 μM) but did slow the on rate of DP178binding (7.9×10⁻³ M⁻¹ s⁻¹ to 3.47×10⁻¹ M⁻¹ s⁻¹) indicating possibly aninduction of protein conformation change (FIGS. 11A, 11B and Table inFIG. 11).

The T649Q26L HR-2 peptide was designed to be of higher affinity forbinding to HR-1 by the Q26L substitution (Shu et al, Biochemistry39:1634-1642 (2000)), and indeed, in this study, T649Q26L also bound toligated, cleaved CD4-gp140 complexes. Soluble CD4 increased the maximalDP178 binding to cleaved gp140 at equilibrium (Rm) by ˜10 fold (from anRm of DP178 binding with no sCD4 of 35 RU (response units) to an Rm of320 RU with sCD4) (FIG. 11). The kd, s⁻¹ (off-rate) of binding for DP178to cleaved gp140 after sCD4 was 0.0056. sCD4 binding to cleaved gp140oligomers resulted in a t½ of DP178 binding of 124 sec. The kd ofbinding for DP178 to sCD4 and CCR5-D1 ligated cleaved gp140 89.6 was 1.1μM. An additional control for the specificity of HR-2 peptide bindingwas the demonstration that the DP178 HR-2 peptide did not bind tosCD4-ligated gp120.

Ability of Biotinylated HR-2 Peptide to Immunoprecipitate HIV EnvelopeProteins. To identify the components in gp140 to which HR-2 peptidesbind in solution, gp140 envelope was immunoprecipitated withbiotinylated DP178 HR-2 peptide, and then the proteins bound tobiotinylated HR-2 were analyzed by Western blot analysis. FIG. 12 showsthe HIV envelope components present in cleaved HIV 89.6 gp140. Lane 10shows gp120 and gp140, immunoblotted with anti-gp120 mab T8. Lane 9shows uncleaved gp140 and gp41 immunoblotted with the anti-gp41 mab,7B2. FIG. 12 also shows that biotinylated HR-2 peptide, DP178,constitutively immunoprecipitated both cleaved gp41 (˜33 kd) anduncleaved gp140 in the absence of sCD4 (lane 3), and the level of gp140and gp41 immunoprecipitated by biotinylated HR-2 was enhanced bysCD4(lane 1). Lanes 5 and 7 show that the anti-gp120 mab T8 recognizedthe gp120 region of the gp140 protein immunoprecipitated by biotinylatedHR-2. Control immunoprecipitations with scrambled biotinylated DP178peptide did not immunoprecipitate significant levels of HIV envelopeproteins (Lanes 2, 4, 6 and 8). CD-4-Induced Binding of HR-2 Peptides toHIV 89.6 gp140 Envelope Proteins. A major goal of this study was todetermine if fusion associated conformations of gp41 could be detectedusing SPR assays of HR-2 peptide binding. It was reasoned that if HR-2peptides can bind gp140, the gp41 coiled-coil structure must beuncoiled, such that endogenous HR-2 is not bound to HR-1. Two such HR-2peptides were used in this study, DP178 (Wild et al, Proc. Natl. Acad.Sci. USA 91:12676-12680 (1994), Rimsky et al, J. Virol. 72:986-993(1998)) and T649Q26L (Rimsky et al, J. Virol. 72:986-993 (1998), (Shu etal, Biochemistry 39:1634-1642 (2000)). DP178 contains C-terminal aminoacids of HR-2 (Wild et al, Proc. Natl. Acad. Sci. USA 91:12676-12680(1994), Rimsky et al, J. Virol. 72:986-993 (1998)) while T649Q26Lcontains more N-terminal amino acids of HR-2 (Rimsky et al, J. Virol.72:986-993 (1998), (Shu et al, Biochemistry 39:1634-1642 (2000)).

HIV envelope gp120 proteins bind to sCD4 with a relatively high affinity(Myszka et al, Proc. Natl. Acad. Sci. USA 97:9026-9031 (2000), Collmanet al, J. Virol. 66:7517-7521 (1992)). In preliminary studies, it wasfound that soluble HIV 89.6 gp120 protein bound strongly to immobilizedsCD4, with a K_(d) 23 nM and with an off-rate of 1.1×10⁻⁴ s¹. Thus, aCD4 immobilized surface allowed a very stable capture of HIV envelope,and this approach has been used to assay HR-2 peptide binding to sCD4bound HIV 89.6 envelope proteins. To create equivalent surfaces oftethered gp140 and gp120 on CM5 sensor chips, sCD4 and anti-gp120 mab T8immobilized on sensor chips were used as capture surfaces. A blank chipserved as an in-line reference surface for subtraction of non-specificbinding and bulk responses. Mab T8 bound HIV 89.6 gp120 with an affinityof 5.6 nM. Thus, both CD4 and the mab T8 provided stable surfaces foranchoring HIV envelope proteins.

Since HIV 89.6 gp140 contains both cleaved and uncleaved gp140, it wasimportant to show that gp41 was present in CD4-gp140 complexes followingcapture of gp140 proteins on CD4 or mab T8 surfaces. When equivalentresponse unit (RU) amounts of gp140 proteins were captured on these twosurfaces (FIG. 14A), the same level of anti-gp120 V3 mab 19b andanti-gp41 mab 2F5 binding was observed (FIGS. 14B and 14C). Mab 2F5reactivity could either be reacting with captured cleaved gp41 orbinding to gp41 in uncleaved gp140. Nonetheless, the captured gp140proteins on both of these surfaces were near identical in theirreactivity with anti-gp120 and anti-gp41 mabs.

The ability of HR-2 peptides to bind to captured gp140 on mab T8 or sCD4surfaces was tested. Binding of the HR-2 peptides showed qualitativedifferences in binding to mab T8 and sCD4-bound gp140. Compared to theT8-gp140 surface (near background binding), the DP178 HR-2 peptide boundspecifically to the sCD4-gp140 surface (FIG. 14D). However, there was nobinding of the scrambled DP178 peptide to mab T8-gp140 or sCD4-gp140surfaces (FIG. 14D). Similar to HR-2 peptide DP178 binding, HR-2 peptideT649Q26L showed no binding to the mab T8-gp140 surface, and markedbinding to the sCD4-gp140 surface (FIG. 14F). Taken together, theseresults demonstrated that sCD4 induced the binding of both HR-2peptides, DP178 and T649Q26L, to HIV 89.6 gp140.

HR-2 peptide binding to CD4-gp140 compared to CD4-gp120 complexes.Kowalski et al. have shown that mutations in gp41 HR-2 disruptgp41-gp120 binding, and that HR-2 contains a touch point site of gp41non-covalent interaction with gp120 (Alam et al, Nature 381:616-620(1996)). Thus, it was of interest to compare HR-2 binding to sCD4-gp140complexes with sCD4-gp120 complexes in SPR assays. As in experiments inFIG. 14, HIV 89.6 gp120 or gp140 proteins were captured in equivalentamounts on sCD4 immobilized surfaces. Interestingly, specific binding ofHR-2 peptide was detected on both sCD4-gp140 and sCD4-gp120 surfaces(FIGS. 15A and 15B). However, there was much higher binding of bothDP178 and T649Q26L HR-2 peptides to the sCD4-gp140 surface compared tothe sCD4-gp120 surface (FIGS. 15A, 15B). To determine if the binding ofHR-2 peptides to gp120 was induced by sCD4, HR-2 binding to sCD4-gp120was compared with HR-2 binding to gp120 proteins captured on the T8 mabsurface. As shown in FIG. 15C, the binding of both HR-2 peptides DP178and T649Q26L to gp120 was specifically induced by sCD4 since neitherHR-2 peptide bound to gp120 on the T8 mab surface. No binding ofscrambled DP178 to CD4-gp140 or CD4-gp120 was detected (FIG. 15D).

Finally, to assess the affinity of the binding interactions between HR-2peptide and sCD4 triggered HIV 89.6 gp140 and gp120, the rate constantswere measured and the dissociation constant (K_(d)) for the binding ofboth HR-2 peptides, DP178 and T649Q26L to sCD-gp140 and sCD4-gp120(FIGS. 15A, 15B, and 15E). There was little difference in the kineticsof HR-2 peptides binding to sCD4-gp140 and sCD4-gp120, indicating thatthe higher level of binding of the HR-2 peptide to CD4-gp140 was due toinduced binding to HR1-gp41 in addition to binding sites on gp120. Thebinding affinity of the HR-2 peptides for gp120 and gp140 were between1.2-2.5 μM. The HR-2 binding was stable with relatively slow off-rates(t_(1/2) values in minutes, 7.7 to 10.5 min) on both sCD4-gp120 andsCD4-gp140 complexes.

Binding of HR-2 peptide to recombinant gp41. To directly determine ifHR-2 peptides can constitutively bind to purified gp41, recombinant ADAgp41 was immobilized to a sensor surface and the binding of HR-2peptide, DP178, determined. The HR-2 peptide DP178 bound well toimmobilized gp41 (FIG. 16A), while no binding was observed with thescrambled DP178 peptide (FIG. 16C). To compare the binding of DP178 HR-2peptide to recombinant gp41 versus HIV 89.6 gp140-sCD4 complex, theequilibrium binding contants (Keq) of HR-2 binding to both weremeasured. While the Keq of DP178 binding to recombinant gp41 was weak at26.7 μM (FIG. 16E), the Keq of DP178 binding to sCD4-gp140 was 10 foldstronger at 1.7 μM (FIG. 16F).

Complexes of gp120-gp41 formed on a sensor surface can be induced bysCD4 to upregulate HR-2 peptide binding. In the preparation of HIV 89.6gp140 envelope, there is uncleaved gp140, and cleaved gp140 componentsof gp120 and gp41. Thus, it is possible that HR-2 peptide could beinduced to bind uncleaved gp140 and/or could be induced to bind tocleaved gp120 and gp41. To directly determine if binding of sCD4 togp120 that is non-covenlently bound to gp41 can upregulate HR-2 peptidebinding to the sCD4-gp120-gp41 complex, recombinant ADA gp41 wasimmobilized on a sensor chip, and HIV 89.6 gp120 or a gp120 sCD4 mixturewas flowed over it. It was found that gp120 bound stably to gp41 (FIG.17). When HR-2 peptide DP178 was flowed over the gp41-gp120-sCD4complex, binding of HR-2 was upregulated compared to HR-2 binding togp41 or to the gp41-gp120 complex (FIG. 17). Thus, sCD4 ligation ofgp120 non-covalently bound to gp41 can upregulate either gp41 or gp120,or both, to bind to HR-2.

Neutralizing Epitopes on HIV 89.6 gp140 Before and After Ligation withsCD4. The 2F5 (anti-gp41, ELDKWAS (SEQ ID NO:5)) (Muster et al, J.Virol. 67:6642-6647 (1993)), mab neutralizes HIV primary isolates. Priorto ligation of cleaved 89.6 gp140 with sCD4, it was found that the 2F5gp41 epitope was exposed. Following sCD4 ligation, the 17b CCR5 bindingsite epitope (2-4) was upregulated and the 2F5 epitope continued to beexpressed.

EXAMPLE 3 Experimental Details

Proteins. Soluble CD4 was produced by Progenics, Tarrytown, N.Y. and wasprovided by the Division of AIDS, NIAID, NIH. Soluble envelope gp120(VBD-2) and gp140 (VBD-1) proteins from HIV 89.6 primary isolate wereproduced using recombinant vaccinia viruses and purified as described(Earl et al, J. Virol. 68:3015-3026 (1994), Earl et al, J. Virol.75:645-653 (2001)). Briefly, BS-C-1 cells in 160 cm² flasks wereinfected with vBD1 (HIV 89.6 gp140) or vBD2 (gp120) viruses. After 2 h,the cells were washed in PBS and placed in serum-free OPTI-MEM media(Gibco) for 24-26 hr. The culture medium was then harvested bycentrifugation and filtration (0.2 μm) and Tritox-X 100 added to 0.5%.For some experiments, culture medium was concentrated 15-30 fold andserved as a source of gp140 glycoproteins (a mix of cleaved anduncleaved forms). Lentil lectin column purified gp140 contained ˜50:50cleaved and uncleaved gp140. Recombinant HIV ADA gp41 protein wasobtained from Immunodiagnostics Inc. (Woburn Mass.). HIV-1 gp120 wasproduced by ABL and provided by the Division of AIDS, NIAID, NIH.

Monoclonal Antibodies. Mab A32 was obtained from James Robinson (TulaneUniversity, New Orleans, La.) (Boots et al, AIDS Res. Hum. Res. 13:1549(1997)). A32 mab was affinity purified from serum-free media using aStaph Protein-G column.

HR-2 Peptides. Synthetic peptides were synthesized (SynPep, Inc.,Dublin, Calif.), and purified by reverse phase HPLC. Peptides used inthis study had greater than 95% purity as determined by HPLC, andconfirmed to be correct by mass spectrometry. gp41 peptides DP178,YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO:2) (Wild et al, Proc.Nati. Acad. Sci. USA 19:12576-12680 (1994)), and T649-Q26L,WMEWDREINNYTSLIHSLIEESQNQLEKNEQELLEL (SEQ ID NO:4) (Rimsky et al, J.Virol. 72:986-993 (1998), Shu et al, Biochemistry 39:1634-1642 (2000))were derived from HIV-1 envelope gp41 from HIV 89.6 (Collman et al, J.Virol. 66:7517-7521 (1992)) . As a control for HR-2 peptide binding, ascrambled sequence DP178 peptide was made. For immunoprecipitations andselect SPR experiments, biotinylated DP178 and DP178 scrambled peptideswere synthesized (SynPep, Inc.).

Surface Plasmon Resonance Biosensor Measurements. SPR biosensormeasurements were determined on a BIAcore 3000 (BIAcore Inc., Uppsala,Sweden) instrument. Anti-gp120 mab (T8) or sCD4 (100-300 μg/ml) in 10 mMNa-Acetate buffer, pH 4.5 were directly immobilized to a CM5 sensor chipusing a standard amine coupling protocol for protein immobilization(Alam et al, Nature 381:616-620 (1996)). A blank in-line referencesurface (activated and de-activated for amine coupling) was used tosubtract non-specific or bulk responses. Binding of proteins andpeptides (biotinylated or free DP178, T649Q26L, DP178-scrambled) wasmonitored in real-time at 25° C. with a continuous flow of PBS (150 mMNaCl, 0.005% surfactant p20), pH 7.4 at 10-30 μl/min. Analyte (proteinsand peptides) were removed and the sensor surfaces were regeneratedfollowing each cycle of binding by single or duplicate 5-10 μl pulses ofregeneration solution (10 mM glycine-HCl, pH 2.5 or 10 mM NaOH).

All analyses were performed using the non-linear fit method ofO'Shannessy et al. (O'Shannessy et al, Anal. Biochem. 205:132-136(1992)) and the BIAevaluation 3.0 software (BIAcore Inc). Rate andequilibrium constants were derived from curve fitting to the Langmuirequation for a simple bimolecular interaction (A+B=AB).

Results

CD4-induced binding of HR-2 Peptides to HIV 89.6 gp140 EnvelopeProteins. A major goal of this study was to determine iffusion-associated conformations of gp41 could be detected using SPRassays of HR-2 peptide binding. It was reasoned that if HR-2 peptidescan bind gp140, the gp41 coiled-coil structure must be uncoiled, suchthat endogenous HR-2 is not bound to HR-1. Two such HR-2 peptides wereused in this study, DP178 (Wild et al, Proc. Natl. Acad. Sci. USA19:12676-12680 (1994), Rimsky et al, J. Virol. 72:986-993 (1998)) andT649Q26L (Rimsky et al, J. Virol. 72:986-993 (1998), Shu et al,Biochemistry 39:1634-1642 (2000)). DP178 contains C-terminal amino acidsof HR-2 (Wild et al, Proc. Natl. Acad. Sci. USA 19:12676-12680 (1994),(Rimsky et al, J. Virol. 72:986-993 (1998)) while T649Q26L contains moreN-terminal amino acids of HR-2 (Rimsky et al, J. Virol. 72:986-993(1998), Shu et al, Biochemistry 39:1634-1642 (2000)).

HIV envelope gp120 proteins bind to sCD4 with a relatively high affinity(Myszka et al, Proc. Natl. Acad. Sci. 97:9026-9031 (2000), Collman etal, J. Virol. 66:7517-7521 (1992)). In preliminary studies, it was foundthat soluble HIV 89.6 gp120 protein bound strongly to immobilized sCD4,with a K_(d) 23 nM and with an off-rate of 1.1×10⁻⁴ s¹. Thus, a CD4immobilized surface allowed a very stable capture of HIV envelope, andthis approach has been used to assay HR-2 peptide binding to sCD4 boundHIV 89.6 envelope proteins. To create equivalent surfaces of tetheredgp140 and gp120 on CM5 sensor chips, sCD4 and anti-gp120 mab T8immobilized on sensor chips were used as capture surfaces. A blank chipserved as an in-line reference surface for subtraction of non-specificbinding and bulk responses. Mab T8 bound HIV 89.6 gp120 with an affinityof 5.6 nM. Thus, both CD4 and the mab T8 provided stable surfaces foranchoring HIV envelope proteins.

Since HIV 89.6 gp140 contains both cleaved and uncleaved gp140, it wasimportant to show that gp41 was present in CD4-gp140 complexes followingcapture of gp140 proteins on CD4 or mab T8 surfaces. When equivalentresponse unit (RU) amounts of gp140 proteins were captured on these twosurfaces, the same level of anti-gp120 V3 mab 19b and anti-gp41 mab 2F5binding was observed. Mab 2F5 reactivity could either be reacting withcaptured cleaved gp41 or binding to gp41 in uncleaved gp140.Nonetheless, the captured gp140 proteins on both of these surfaces werenear identical in their reactivity with anti-gp120 and anti-gp41 mabs.

The ability of HR-2 peptides to bind to captured gp140 on mab T8 or sCD4surfaces was next tested. Binding of the HR-2 peptides showedqualitative differences in binding to mab T8 and sCD4-bound gp140.Compared to the T8-gp140 surface (near background binding), the DP178HR-2 peptide bound specifically to the sCD4-gp140 surface. However,there was no binding of the scrambled DP178 peptide to mab T8-gp140 orsCD4-gp140 surfaces. Similar to HR-2 peptide DP178 binding, HR-2 peptideT649Q26L showed no binding to the mab T8-gp140 surface, and markedbinding to the sCD4-gp140 surface. Taken together, these resultsdemonstrated that sCD4 induced the binding of both HR-2 peptides, DP178and T649Q26L, to HIV 89.6 gp140.

The A32 mab has been reported to reproduce the effect of sCD4 intriggering HIV envelope to upregulated the availability of CCR5 bindingsite (Wyatt et al, J. Virol 69:5723 (1995)). Thus, an A32 mab surfacewas used to determine if A32 mab could mimic sCD4 to upregulate HR-2binding to captured gp140. Similar to sCD4, HR-2 peptide binding wasmarkedly upregulated when gp140 (a mixture of uncleaved gp140, cleavedgp120 and cleaved g41) was captured on the A32 mab surface compared togp140 captured on the mab T8 surface (FIG. 26). Similar results wereobtained using A32 mab and gp120.

All documents cited above are hereby incorporated in their entirety byreference.

One skilled in the art will appreciate from a reading of this disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention.

1. An isolated immunogen comprising an HIV envelope protein bound to aligand, which ligand upregulates at least one of the CD4 binding siteand the CCR5 binding site on said protein, wherein said protein is gp120noncovalently bound to gp41, and wherein said immunogen furthercomprises an HR-2 peptide bound to said protein.
 2. The immunogenaccording to claim 1 wherein said ligand is an antibody, or Fab2 or Fabfragment thereof.
 3. The immunogen according to claim 1 wherein saidligand binds to a CCR₅ binding site on gp120 and upregulates a CD4binding site on gp120.
 4. The immunogen according to claim 3 whereinsaid ligand is an antibody, or Fab₂ or Fab fragment thereof.
 5. Theimmunogen according to claim 3 wherein said ligand is monoclonalantibody (mab) 17b, or Fab₂ or Fab fragment thereof, or mimic thereof.6. The immunogen according to claim 1 wherein said ligand upregulates aCCR5 and a CD4 binding site on gp120.
 7. The immunogen according toclaim 6 wherein said ligand is an antibody, or Fab₂ or Fab fragmentthereof.
 8. The immunogen according to claim 6 wherein said ligand bindsto a site on gp120 to which mab A32 binds.
 9. The immunogen according toclaim 8 wherein said ligand is mab A32, or Fab₂ or Fab fragment thereof,or mimic thereof.
 10. The immunogen according to claim 1 wherein saidprotein is in soluble form.
 11. The immunogen according to claim 1wherein said protein is associated with a cell vesicle or liposome. 12.The immunogen according to claim 1 wherein gp120, gp41 and said ligandare crosslinked.
 13. The immunogen according to claim 1 wherein gp120,gp41, said ligand and said HR-2 peptide are crosslinked.